Field of the Invention
The present invention relates to a method for quantifying a target nucleic acid sequence by use of a melting analysis.
Description of the Related Art
A target nucleic acid amplification process is prevalently involved in most of technologies for detecting target nucleic acid sequences. Nucleic acid amplification is a pivotal process for a wide variety of methods in molecular biology, such that various amplification methods have been proposed.
The most predominant process for nucleic acid amplification known as polymerase chain reaction (hereinafter referred to as “PCR”) is based on repeated cycles of denaturation of double-stranded DNA, followed by oligonucleotide primer annealing to the DNA template, and primer extension by a DNA polymerase (Mullis et al. U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159; Saiki et al., (1985) Science 230, 1350-1354).
As alternatives, various methods such as LCR (Ligase Chain Reaction), DA (Strand Displacement Amplification), NASBA (Nucleic Acid Sequence-Based Amplification), TMA (Transcription Mediated Amplification) and RCA (Rolling-Circle Amplification) have been suggested.
As application of nucleic acid amplification to target detection, the real-time detection methods are widely utilized to detect a target sequence with measuring nucleic acid amplification in a real-time manner.
The real-time detection methods generally use labeled probes specifically hybridized with target sequences. As examples of methods using hybridization between labeled probes and target sequences, the Molecular beacon method using dual-labeled probes capable of hairpin structure (Tyagi et al, Nature Biotechnology v. 14 Mar. 1996), the Hybridization probe method using two probes singly labeled with donor or acceptor (Bernad et al, 147-148 Clin Chem 2000; 46) and the Lux method using single-labeled oligonucleotides (U.S. Pat. No. 7,537,886) have been developed. In addition, the TaqMan method using cleavage reaction of a dual-labeled probe by the 5′-nuclease activity of DNA polymerases as well as hybridization of dual-labeled probes has been widely employed (U.S. Pat. Nos. 5,210,015 and 5,538,848).
In the real-time detection methods as homogenous assay, the amplification and detection analysis are performed in a single tube such their performance is relatively convenient. In addition, the real-time detection methods are free from contaminations. However, for simultaneous detection of a plurality of target sequences in a real-time manner, there is limitation not only in utilization fluorescent molecules due to assignment of a fluorescent molecule to a target nucleic acid molecule but also in detectable target number per sample due to spectrum interference between fluorescent molecules.
As alternatives for target detection in a homogenous assay, the post-PCR melting assay has been suggested in which fluorescent intensities are monitored with increasing or decreasing in a temperature range after target amplification and then amplicons are detected using the melting profile.
U.S. Pat. No. 5,871,908 and U.S. Pat. No. 6,174,670 disclose target detection methods by melting analysis of target sequences in a double strand or duplex between target sequence and probe. WO 2012/096523 discloses a melting analysis of extended duplexes formed dependent on the presence of target sequences. This method that does not use target sequences for melting analysis forms extended duplexes having pre-selected sequences when the target sequence is present, which renders a melting analysis to be performed in more effective manner.
Because the methods using a melting analysis utilizes melting temperatures particular to duplexes, they have advantages in which a plurality of target sequences are simultaneously detected by use of a single type of fluorescent label (see U.S. Pat. No. 8,039,215).
In the meantime, the quantification of target sequences as well as target detection is usually required for prognosis determination and analysis of drug responsiveness in the diagnostic field.
The real-time PCR assay is used to quantify target sequences by use of standard quantification curve and Ct (threshold cycle) value. The amplification curves are obtained for standard materials prepared by serious dilution of target sequences with known concentration, and the standard quantification curve is then plotted using the log values of initial amounts of standard materials and Ct values. The Ct value of unknown sample is obtained by real-time PCR and then quantified using the standard quantification curve. Although the quantification method is relatively convenient, it has serious problems due to loss in sample extraction step and PCR inhibition. For overcoming such problems, a quantification method using internal control has been suggested.
The post-PCR melting assay has been applied to quantification by use of height or area of melting peaks.
For example, U.S. Pat. No. 6,245,514 describes that a target sequence and a nucleic acid molecule with known concentration (reference template) are amplified in the presence of nucleic-acid-binding fluorescent dye, melting peaks for amplification products are obtained and integrated to determine relative amounts of the target sequence and the reference template, thereby calculating concentration of the target sequence. The target sequence and the reference template can be differentially detected because they have different Tm values from each other. U.S. Pat. No. 6,174,670 discloses a quantification method using hybridization probes and melting peaks. Since the methods described above performs a melting analysis after target amplification, they have disadvantages in that quantification results are likely to be varied depending on the cycle of amplification termination and the cycle for melting analysis.
U.S. Pat. No. 8,039,215 discloses that a melting analysis in target amplification is performed to obtain the maximum values of melting peaks for each amplification cycle and a cycle reaching to the maximum value over a threshold value is determined, thereby quantifying a target sequence. Because this method requires melting analyses for most of amplification cycles to determine a cycle reaching to the maximum value over a threshold value, it demands longer analysis time.
Under such circumstances of conventional technologies, the present inventor has recognized that a plurality of target sequences can be simultaneously detected and quantified in a more effective manner, when a quantification method using a melting analysis for detection of a plurality of target sequences with a single detecting channel and being capable for providing improved quantification results in more rapid manner is developed.
Throughout this application, various patents and publications are referenced and citations are provided in parentheses. The disclosure of these patents and publications in their entities are hereby incorporated by references into this application in order to more fully describe this invention and the state of the art to which this invention pertains.